N-aryl pyrrolidine derivatives as beta-secretase inhibitors

ABSTRACT

There is provided a series of substituted N-aryl pyrrolidine derivatives of Formula (I) 
                         
or a stereoisomer; or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 5′ , R 6 , R 7 , and p as defined herein, their pharmaceutical compositions and methods of use. These compounds inhibit the processing of amyloid precursor protein (APP) by β-secretase and, more specifically, inhibit the production of Aβ-peptide. The present disclosure is directed to compounds useful in the treatment of neurological disorders related to β-amyloid production, such as Alzheimer&#39;s disease and other conditions affected by anti-amyloid activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a non-provisional application which claims the benefit of U.S.Provisional Application No. 60/788,844 filed Apr. 3, 2006.

FIELD OF THE DISCLOSURE

This patent application provides N-aryl pyrrolidine derivatives havingdrug and bio-affecting properties, their pharmaceutical compositions andmethod of use. In particular, the disclosure is concerned with a seriesof N-aryl pyrrolidine derivatives which are inhibitors of β-amyloidpeptide (β-AP) production, thereby acting to prevent the accumulation ofamyloid protein deposits in the brain and, therefore, are useful in thetreatment of neurological disorders related to β-amyloid production.More particularly, the present disclosure relates to the treatment ofAlzheimer's Disease (AD) and similar diseases.

BACKGROUND

Alzheimer's Disease is a progressive, neurodegenerative disordercharacterized by memory impairment and cognitive dysfunction. AD ischaracterized pathologically by the accumulation of senile (neuritic)plaques, neurofibrillary tangles, amyloid deposition in neural tissuesand vessels, synaptic loss, and neuronal death. It is the most commonform of dementia and it now represents the third leading cause of deathafter cardiovascular disorders and cancer. The cost of Alzheimer'sDisease is enormous (in the U.S., greater than $100 billion annually)and includes the suffering of the patients, the suffering of families,and the lost productivity of patients and caregivers. As the longevityof society increases, the occurrence of AD will markedly increase. It isestimated that more than 10 million Americans will suffer from AD by theyear 2020, if methods for prevention and treatment are not found.Currently, AD is estimated to afflict 10% of the population over age 65and up to 50% of those over the age of 85. No treatment that effectivelyprevents AD or reverses the clinical symptoms and underlyingpathophysiology is currently available (for review, see Selkoe, D. J.Ann. Rev. Cell Biol., 1994, 10: 373-403).

Histopathological examination of brain tissue derived upon autopsy orfrom neurosurgical specimens in affected individuals reveals theoccurrence of amyloid plaques and neurofibrillar tangles in the cerebralcortex of such patients. Similar alterations are observed in patientswith Trisomy 21 (Down's syndrome). Biochemical and immunological studiesreveal that the dominant proteinaceous component of the amyloid plaqueis an approximately 4.2 kilodalton (kD) protein of about 39 to 43 aminoacids. This protein is designated Aβ, β-amyloid peptide, and sometimesβ/A4; referred to herein as Aβ. In addition to its deposition in amyloidplaques, Aβ is also found in the walls of meningeal and parenchymalarterioles, small arteries, capillaries, and sometimes, venules.Compelling evidence accumulated during the last decade reveals that Aβis an internal polypeptide derived from a type 1 integral membraneprotein, termed β-amyloid precursor protein (APP) (Selkoe, D. Physiol.Rev. 2001, 81, 741-766; Wolfe, M. J. Med. Chem. 2001, 44, 2039-2060).βAPP is normally produced by many cells both in vivo and in culturedcells, derived from various animals and humans. Several proteolyticfragments of APP are generated by proteinases referred to as secretases.A subset of these proteolytic fragments, designated β-amyloid peptide(Aβ), contains 39 to 43 amino acids and is generated by the combinedaction of β-secretase and γ-secretase. β-secretase is a membrane-bound,aspartyl protease that forms the N-terminus of the Aβ peptide. TheC-terminus of the Aβ peptide is formed by γ-secretase, an apparentlyoligomeric complex that includes presenilin-1 and/or presenilin-2.Presenilin-1 and presenilin-2 are polytopic membrane-spanning proteinsthat may contain the catalytic components of γ-secretase (Seiffert, D.;Bradley, J. et al., J. Biol. Chem. 2000, 275, 34086-34091).

In addition to AD, excess production and/or reduced clearance of Aβcauses cerebral amyloid angiopathy (CAA) (reviewed in Thal, D.,Gherbremedhin, E. et al., J. Neuropath. Exp. Neuro. 2002, 61, 282-293).In these patients, vascular amyloid deposits cause degeneration ofvessel walls and aneurysms that may be responsible for 10-15%hemorrhagic strokes in elderly patients. As in AD, mutations in the geneencoding Aβ lead to an early onset form of CAA, referred to as cerebralhemorrhage with amyloidosis of the Dutch type, and mice expressing thismutant protein develop CAA that is similar to patients.

A logical approach to reducing Aβ levels is to interfere with the actionof the secretases that are directly involved in the cleavage of APP toAβ. The β-secretase enzyme (BACE) is responsible for cleaving APP andforms the amino-terminus of Aβ, initiating the amyloidogenic pathway.The BACE enzyme is a transmembrane aspartyl protease and was describedin the literature by several independent groups [see Hussain, I. et al.,(1999) Mol. Cell. Neurosci., 14: 419-427; Lin, X. et al., (2000)Proceedings of the National Academy of Sciences of the United States ofAmerica, 97: 1456-1460; Sinha, S., et al., (1999) Nature (London), 402:537-540; Vassar, R., et al., (1999) Science (Washington, D.C.), 286:735-741; Walsh, D. M. et al., (2002); Wolfe, M. S. (2001); Yan, R. etal., (1999) Nature (London), 402: 533-537].

Removal of BACE activity in mice by gene targeting completely abolishesAβ production [see Luo, Y., et al., (2001) Nature Neuroscience, 4:231-232; Roberds, S. L. et al., (2001) Human Molecular Genetics, 10:1317-1324].

BACE −/− mice also show no detectable negative phenotypes, suggestingthat disruption of BACE-mediated cleavage of APP does not produceadditional undesired effects. This demonstrates that a drug substancecapable of inhibiting β-secretase activity should lower or halt thesynthesis of Aβ and should provide a safe treatment for Alzheimer'sdisease.

At present there remains an urgent need to develop pharmaceutical agentscapable for effective treatment in halting, slowing, preventing, and/orreversing the progression of Alzheimer's disease. Compounds that areeffective inhibitors of beta-secretase, that inhibit beta-secretasemediated cleavage of APP, that are effective inhibitors of Aβ proteinproduction by beta-secretase, and/or are effective in reducing solubleAβ protein, amyloid beta deposits or amyloid beta plaques, are neededfor effective treatment in halting, slowing, preventing, and/orreversing neurological disorders related to Aβ protein production, suchas Alzheimer's disease.

SUMMARY OF THE DISCLOSURE

A series of N-aryl pyrrolidine derivatives having the Formula (I)

or a stereoisomer; or a pharmaceutically acceptable salt thereof,wherein R₁, R₂, R₃, R₄, R₅, R_(5′), R₆, R₇ and p as defined below areeffective inhibitors of the production of β-amyloid peptide (β-AP) fromβ-amyloid precursor protein (β-APP). The pharmacologic action of thesecompounds makes them useful for treating conditions responsive to theinhibition of β-AP in a patient; e.g., Alzheimer's Disease (AD) andDown's Syndrome. Therapy utilizing administration of these compounds ora pharmaceutical composition containing a therapeutically effectiveamount of at least one of these compounds to patients suffering from, orsusceptible to, these conditions involves reducing β-AP available foraccumulation and deposition in brains of these patients.

DETAILED DESCRIPTION

The present application comprises compounds of Formula I, theirpharmaceutical formulations, and their use in inhibiting β-AP productionin patients suffering from or susceptible to AD or other disordersresulting from β-AP accumulation in brain tissue. The compounds ofFormula I which include stereoisomers and pharmaceutically acceptablesalts thereof have the following formula and meanings:

wherein

-   R₁ is phenyl optionally substituted with one or more groups selected    from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy,    C₂₋₆alkenyl, C₁₋₆alkyl optionally substituted with OH,    C₃₋₆cycloalkyl, or —NH₂, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl,    —(CH₂)_(m)—NHC(═O)Ophenyl optionally substituted with halogen,    —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉;-   R₂ and R₃ are each independently hydrogen, methyl or hydroxymethyl;-   p is 0 or 1;-   m is 0 or 1;-   R₄ and R₆ are independently hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH,    NH₂, benzyloxy, phenyl or CF₃ in which said phenyl is optionally    substituted with one or more groups selected from halogen, CN, CF₃    and OCH₃;-   R₅ and R_(5′) are each independently hydrogen, C₁₋₆alkyl, or    C₁₋₄alkanol;-   R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂,    N(CH₃)₂, NO₂, or CF₃;-   R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally    substituted with a group selected from halogen, CN, CF₃ and    C₁₋₄alkoxy;-   R₉ is —C₁₋₆alkylNR₁₀R₁₁;-   R₁₀ is hydrogen or C₁₋₆alkyl; and-   R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted with OH, halogen,    C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl,    —(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl or    C₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen,    hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with the    nitrogen to which they are attached is azetidine, aziridine,    pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine    or thiomorpholine, in which each is optionally substituted with a    group selected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy;    or a nontoxic pharmaceutically acceptable salt thereof.

The present application also provides a method for the treatment oralleviation of disorders associated with β-amyloid peptide, especiallyAlzheimer's Disease, cerebral amyloid angiopathy and Down's Syndrome,which comprises administering together with a conventional adjuvant,carrier or diluent a therapeutically effective amount of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof.

As used herein, the term “Aβ” denotes the protein designated Aβ,β-amyloid peptide, and sometimes β/A4, in the art. Aβ is anapproximately 4.2 kilodalton (kD) protein of about 39 to 43 amino acidsfound in amyloid plaques, the walls of meningeal and parenchymalarterioles, small arteries, capillaries, and sometimes, venules. Theisolation and sequence data for the first 28 amino acids are describedin U.S. Pat. No. 4,666,829. The 43 amino acid sequence is well known inthe art, see Colin Dingwall, Journal of Clinical Investigation, November2001, 108 (9): 1243-1246; as well as PCT international patentapplication WO 01/92235, published Dec. 6, 2001, herein incorporated byreference in its entirety.

The term “APP”, as used herein, refers to the protein known in the artas β amyloid precursor protein. This protein is the precursor for Aβ andthrough the activity of “secretase” enzymes, as used herein, it isprocessed into Aβ. Differing secretase enzymes, known in the art, havebeen designated β secretase, generating the N-terminus of Aβ, αsecretase cleaving around the 16/17 peptide bond in Aβ, and “γsecretases”, as used herein, generating C-terminal Aβ fragments endingat position 38, 39, 40, 42, and 43 or generating C-terminal extendedprecursors which are subsequently truncated to the above polypeptides.

The term “substituted,” as used herein and in the claims, means that anyone or more hydrogens on the designated atom is replaced with aselection from the indicated group, provided that the designated atom'snormal valency is not exceeded, and that the substitution results in astable compound.

As used herein and in the claims, “alkyl” or “alkylene” is intended toinclude both branched and straight-chain saturated aliphatic hydrocarbongroups having the specified number of carbon atoms; for example, “C₁₋₆alkyl” denotes alkyl having 1 to 6 carbon atoms. Examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, i-propyl,n-butyl, i-butyl, sec-butyl, t-butyl, pentyl, 3-methylbutyl, hexyl andthe like. Preferred “alkyl” group, unless otherwise specified, is “C₁₋₄alkyl”. Additionally, unless otherwise specified, “propyl” denotesn-propyl or i-propyl; “butyl” denotes n-butyl, i-butyl, sec-butyl, ort-butyl.

As used herein and in the claims, “alkanol” is intended to include bothbranched and straight-chain saturated aliphatic hydrocarbon groupsbearing a hydroxyl group having the specified number of carbon atoms;for example, “C₁₋₆ alkanol” denotes alkanol having 1 to 6 carbon atoms.Examples of alkyl include, but are not limited to, hydroxymethyl,hydroxyethyl, 3-hydroxy-n-propyl, 2-hydroxy-i-propyl, 4-hydroxy-n-butyl,and the like. Preferred “alkanol” group, unless otherwise specified, is“C₁₋₄ alkanol”.

As used herein and in the claims, “alkenyl” or “alkenylene” is intendedto include hydrocarbon chains of either a straight or branchedconfiguration and one or more unsaturated carbon-carbon bonds which mayoccur in any stable point along the chain, for example, “C₂₋₆ alkenyl”include but are not limited to ethenyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 2-pentenyl,3-pentenyl, hexenyl, and the like.

As used herein and in the claims, “alkynyl” or “alkynylene” is intendedto include hydrocarbon chains of either a straight or branchedconfiguration and one or more carbon-carbon triple bonds which may occurin any stable point along the chain, for example, “C₂₋₆ alkynyl” includebut not limited to ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, and the like.

“Alkoxy” or “alkyloxy” represents an alkyl group as defined above withthe indicated number of carbon atoms attached through an oxygen bridge.Examples of “C₁₋₆alkoxy” include, but are not limited to, methoxy,ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy,and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, s-butoxy, t-butoxy.

As used herein and in the claims, “halogen” refers to fluoro, chloro,bromo, and iodo. Unless otherwise specified, preferred halogens arefluoro and chloro. “Counterion” is used to represent a small, negativelycharged species such as chloride, bromide, hydroxide, acetate, sulfate,and the like.

“Cycloalkyl” is intended to include saturated ring groups, having thespecified number of carbon atoms. For example, “C₃₋₆ cycloalkyl” denotessuch as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The compounds described herein may have asymmetric centers. It isunderstood, that whether a chiral center in an isomer is “R” or “S”depends on the chemical nature of the substituents of the chiral center.All configurations of compounds of the invention are considered part ofthe invention. Additionally, the carbon atom to which R₂ and R₃ isattached and carbon atoms of the pyrrolidine ring may describe a chiralcarbon. Compounds of the present disclosure containing an asymmetricallysubstituted atom may be isolated in optically active or racemic forms.It is well known in the art how to prepare optically active forms, suchas by resolution of racemic forms or by synthesis from optically activestarting materials. Mixtures of isomers of the compounds of the examplesor chiral precursors thereof can be separated into individual isomersaccording to methods which are known per se, e.g. fractionalcrystallization, adsorption chromatography or other suitable separationprocesses. Resulting racemates can be separated into antipodes in theusual manner after introduction of suitable salt-forming groupings, e.g.by forming a mixture of diastereosiomeric salts with optically activesalt-forming agents, separating the mixture into diastereomeric saltsand converting the separated salts into the free compounds. Theenantiomeric forms may also be separated by fractionation through chiralhigh pressure liquid chromatography columns. Many geometric isomers ofolefins and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentinvention. Cis and trans geometric isomers of the compounds of thepresent invention are described and may be isolated as a mixture ofisomers or as separated isomeric forms. All chiral, diastereomeric,racemic forms and all geometric isomeric forms of a structure areintended, unless the specific stereochemistry or isomeric form isspecifically indicated.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein and in the claims, “pharmaceutically acceptable salts”refer to derivatives of the disclosed compounds wherein the parentcompound is modified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the present application can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

In the method of the present application, the term “therapeuticallyeffective amount” means the total amount of each active component of themethod that is sufficient to show a meaningful patient benefit, i.e.,healing of acute conditions characterized by inhibition of β-amyloidpeptide production. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously. The terms“treat, treating, treatment” as used herein and in the claims meanspreventing or ameliorating diseases associated with β-amyloid peptide.

The compounds of the present application can be prepared in a number ofways well known to one skilled in the art of organic synthesis. Thecompounds of the present application can be synthesized using themethods described below, together with synthetic methods known in theart of synthetic organic chemistry, or variations thereon as appreciatedby those skilled in the art. Preferred methods include, but are notlimited to, those described below. All references cited herein arehereby incorporated in their entirety herein by reference.

The compounds may be prepared using the reactions and techniquesdescribed in this section. The reactions are performed in solventsappropriate to the reagents and materials employed and are suitable forthe transformations being effected. Also, in the description of thesynthetic methods described below, it is to be understood that allproposed reaction conditions, including choice of solvent, reactionatmosphere, reaction temperature, duration of the experiment and workupprocedures, are chosen to be the conditions standard for that reaction,which should be readily recognized by one skilled in the art. It isunderstood by one skilled in the art of organic synthesis that thefunctionality present on various portions of the molecule must becompatible with the reagents and reactions proposed. Such restrictionsto the substituents which are compatible with the reaction conditionswill be readily apparent to one skilled in the art and alternate methodsmust then be used.

Compounds of formula I are prepared as illustrated in Scheme 1.Treatment of the appropriate pyrrolidine derivative (II) with an arylhalide (bromide or iodide are preferred) in the presence of CuO atelevated temperatures affords the N-aryl intermediates of formula III.Amide bond coupling of III with N-Boc-S-Methylisothiourea affordscompounds IV. Intermediates of formula IV are then combined with aminesV in the presence of organic base to afford the penultimate compoundsVI. Intermediates of formula V can be obtained commercially, can beprepared by methods known in the literature, or can be readily preparedby one skilled in the art. Treatment of intermediates of formula VI withtrifluoroacetic acid (TFA) provides compounds of formula Ia.

Compounds of formula II are generally known in the literature and alarge number of these derivatives are available to one skilled in theart. Representative procedures for forming substituted pyrrolidines canbe found in many reviews, including Coldham, I. and Hufron, R.“Intramolecular dipolar cycloaddition reactions of azomethine ylides”Chemical Reviews, 2005, 105 (7), 2765-2809.

A particularly useful reaction in this regard is the 2+3 cycloadditionof azomethine ylides. One variant of this reaction is shown in Scheme 2(Philip Garner and Uemit Kaniskan, Tetrahderon Letters, 2005, 46 (31),5181), and there are many additional types known in the literature andaccessible to one skilled in the art.

Additionally, compounds of formula Ib wherein R represents hydrogen orone or more substituents selected from the groups defined herein areprepared by further modifying intermediates of formula VIa where ananiline functionality is present. For instance (Scheme 3), intermediateVIa is sequentially treated with bromoacetyl bromide to form thebromomethyl amide, then with an appropriate amine to afford theintermediate VII. Appropriate amines can be obtained commercially, canbe prepared by methods known in the literature, or can be readilyprepared by one skilled in the art. Deprotection with TFA then affordscompounds Ib.

Compounds of formula Va are prepared from intermediates of formula VIIIas outlined in Scheme 4. Treatment of intermediates of formula VIII withN-bromosuccinimide in the presence of a radical initiator (AIBN orbenzoylperoxide) provides intermediates of formula IX. Intermediates offormula VIII can be obtained commercially, can be prepared by methodsknown in the literature, or can be readily prepared by one skilled inthe art. Intermediates of formula IX are converted to compounds offormula Va via two routes. In the first route, intermediates of formulaIX are treated with sodium azide to provide intermediates of formula X,which upon treatment with lithium aluminum hydride affords compounds offormula Va. Alternatively, intermediates of formula IX are treated withthe potassium salt of phthalimide to provide intermediates of formulaXI, which upon treatment with hydrazine affords compounds of formula Va.

Compounds of formula Vb are also prepared from nitrile-containingintermediates of formula XII as outlined in Scheme 5. Treatment ofintermediates of formula XII with strong hydride-based reducing agentssuch as lithium aluminum hydride or borane provided compounds of formulaVb.

Compounds of formula Vb are also prepared from carboxylic acidderivatives of formula XIII as outlined in Scheme 6. Treatment ofintermediates of formula XIII with ammonia under typical amide bondformation conditions affords intermediates of formula XIV, which upontreatment with strong hydride-based reducing agents such as boraneprovides compounds of formula Vb.

In a preferred, the present invention includes compounds of Formula (II)or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy,C₂₋₆alkenyl, C₁₋₆alkyl optionally substituted with OH, C₃₋₆cycloalkyl,or —NH₂, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl, —(CH₂)_(m)—NHC(═O)Ophenyloptionally substituted with halogen, —(CH₂)_(m)—NHC(═O)R₈ and—NHC(═O)R₉; p is 0 or 1; m is 0 or 1; R₄ is hydrogen, C₁₋₆alkyl,C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenylis optionally substituted with one or more groups selected from halogen,CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN,OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl inwhich each is optionally substituted with a group selected from halogen,CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen orC₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted withOH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl,—(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl orC₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen,hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with thenitrogen to which they are attached is azetidine, aziridine,pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine orthiomorpholine, in which each is optionally substituted with a groupselected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; or a nontoxicpharmaceutically acceptable salt thereof.

In another preferred embodiment, the present invention includescompounds of Formula (III) or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substitutedwith cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉;R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl orCF₃ in which said phenyl is optionally substituted with one or moregroups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen,C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ isC₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted witha group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is—C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen,C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy,C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyloptionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyloptionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy;or R₁₀ and R₁₁ together with the nitrogen to which they are attached isazetidine, aziridine, pyrrolidine, piperidine, homopiperidine,homopiperazine, morpholine or thiomorpholine, in which each isoptionally substituted with a group selected from halogen, C₁₋₆alkyl andC₁₋₄alkoxy; m is 0 or 1; or a nontoxic pharmaceutically acceptable saltthereof.

In still another preferred embodiment, the present invention includescompounds of Formula (IV)

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substitutedwith cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉;R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl orCF₃ in which said phenyl is optionally substituted with one or moregroups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen,C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ isC₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted witha group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is—C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen,C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy,C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyloptionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyloptionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy;or R₁₀ and R₁₁ together with the nitrogen to which they are attached isazetidine, aziridine, pyrrolidine, piperidine, homopiperidine,homopiperazine, morpholine or thiomorpholine, in which each isoptionally substituted with a group selected from halogen, C₁₋₆alkyl andC₁₋₄alkoxy; m is 0 or 1; or a nontoxic pharmaceutically acceptable saltthereof.

In yet another preferred embodiment, the present invention includescompounds of Formula (IV)

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substitutedwith cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉;R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, benzyloxy, phenyl or CF₃ in whichsaid phenyl is optionally substituted with one or more groups selectedfrom halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl,C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl orC₃₋₆cycloalkyl in which each is optionally substituted with a groupselected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁;R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionallysubstituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl,—(CH₂)_(m)C₃₋₆cycloalkyl; —(CH₂)_(m)phenyl optionally substituted withhalogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together withthe nitrogen to which they are attached is azetidine, aziridine,pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine orthiomorpholine; m is 0 or 1; or a nontoxic pharmaceutically acceptablesalt thereof.

In a further embodiment, this invention includes pharmaceuticalcompositions comprising at least one compound of Formula I incombination with a pharmaceutical adjuvant, carrier or diluent.

In another further embodiment, this invention relates to a method oftreatment or prevention of disorders responsive to the inhibition ofβ-amyloid peptide in a mammal in need thereof, which comprisesadministering to said mammal a therapeutically effective amount of acompound of Formula I or a nontoxic pharmaceutically acceptable salt.

In yet another further embodiment, this invention relates to a methodfor treating Alzheimer's Disease, cerebral amyloid angiopathy and Down'sSyndrome in a mammal in need thereof, which comprises administering tosaid mammal a therapeutically effective amount of a compound of FormulaI or a non-toxic pharmaceutically acceptable salt.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The compounds of this application and their preparation can beunderstood further by the following working examples. These examples aremeant to be illustrative of the present application, and are not to betaken as limiting thereof.

Chemical abbreviations used in the specification and Examples aredefined as follows:

-   “Ac” for acetate,-   “APCI” for atmospheric pressure chemical ionization,-   “Boc” or “BOC” for t-butyloxycarbonyl,-   “BOP” for benzotriazol-1-yloxytris-(dimethylamino)-phosphonium    hexafluorophosphate,-   “Cbz” for benzyloxycarbonyl,-   “CDI” for 1,1′-carbonyldiimidazole,-   “CD₃OD” for deuteromethanol,-   “CDCl₃” for deuterochloroform,-   “DCC” for 1,3-dicyclohexylcarbodiimide,-   “DCM” for dichloromethane-   “DEAD” for diethyl azodicarboxylate,-   “DIEA”, “Hunig's base”, or “DIPEA” for N,N-diisopropylethylamine,-   “DMF” for N,N-dimethylformamide,-   “DMAP” for 4-dimethylaminopyridine,-   “DMPU” for 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidone,-   “DMSO” for dimethylsulfoxide,-   “DPPA” for diphenylphosphorylazide-   “EDC” or “EDCI” for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide    hydrochloride,-   “Et” for ethyl,-   “EtOAC” for ethyl acetate,-   “HOAc” for acetic acid,-   “HOBt” for 1-hydroxybenzotriazole hydrate,-   “HATU” for O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate,-   “HMPA” for hexamethylphosphoramide,-   “LDA” for lithium diisopropylamide,-   “LiHMDS” for lithium bis(trimethylsilyl)amide,-   “NaHMDS” for sodium bis(trimethylsilyl)amide,-   “NMM” for 4-methylmorpholine,-   “PyBOP” for benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate,-   “TMSCH₂N₂” for (trimethylsilyl)diazomethane,-   “TMSN₃” for Azidotrimethylsilane,-   “TBTU” for O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    tetrafluoroborate,-   “TEA” for triethylamine,-   “TFA” for trifluoroacetic acid, and-   “THF” for tetrahydrofuran.

Abbreviations used in the Examples are defined as follows: “° C.” fordegrees Celsius, “MS” for mass spectrometry, “ESI” for electrosprayionization mass spectroscopy, “HR” for high resolution, “LC-MS” forliquid chromatography mass spectrometry, “eq” for equivalent orequivalents, “g” for gram or grams, “h” for hour or hours, “mg” formilligram or milligrams, “mL” for milliliter or milliliters, “mmol” formillimolar, “M” for molar, “min” for minute or minutes, “rt” for roomtemperature, “NMR” for nuclear magnetic resonance spectroscopy, “tlc”for thin layer chromatography, “atm” for atmosphere, and “α”, “β”, “R”,“S”, “E”, and “Z” are stereochemical designations familiar to oneskilled in the art.

“HPLC” is an abbreviation used herein for high pressure liquidchromatography. Reverse-phase HPLC can be carried out using a variety ofcolumns with gradient elution from 0% to 100% buffer B in buffer A(buffer A: 90% water and 10% MeOH containing 0.1% trifluoroacetic acid,buffer B: 10% water, 90% MeOH containing 0.1% trifluoroacetic acid). Ifnecessary, organic layers can be dried over sodium sulfate unlessotherwise indicated. However, unless otherwise indicated, the followingconditions are generally applicable. “LC-MS” refers to high pressureliquid chromatography carried out according to the definition for HPLCwith a mass spectrometry detector.

Melting points were determined on a Mel-Temp II apparatus and areuncorrected. IR spectra were obtained on a single-beam Nicolet NexusFT-IR spectrometer using 16 accumulations at a resolution of 4.00 cm−1on samples prepared in a pressed disc of KBr or as a film on KBr plates.Proton NMR spectra (300 MHz, referenced to tetramethylsilane) wereobtained on a Varian INOUA 300, Bruker Avance 300, Avance 400, or Avance500 spectrometer. Data were referred to the lock solvent. ElectrosprayIonization (ESI) experiments were performed on a Micromass II Platformsingle-quadrupole mass spectrometer, or on a Finnigan SSQ7000 massspectrometer. HPLC analyses were obtained using the following columnswith UV detection at 223 nm using a standard solvent gradient program asfollows:

1 minute at 100% buffer A, followed by a gradient from 100% buffer A to100% buffer B over 1-3 minutes, followed by 1 minute at 100% buffer B.The length of the gradient can be determined by the total run time less2 minutes. Retention times (RT) and Run times (RunT) are reported asRT/RunT in the experimental section. The columns employed include:

Column 1: XTerra 4.6×30 mm S5

Column 2: Phenomenex-Luna 4.6×50 mm S10

Column 3: Phenomenex-Luna 4.6×30 mm S10

Prep HPLC solvent conditions: When described as performed under“standard conditions”, Samples were dissolved in acetonitrile (up to 100mg/mL) and run using the following gradient program with a solvent flowrate of 40.0 mL/min on an Xterra prep 18 column.

Acetonitrile H₂O Time (min) (0.05% TFA) (0.05% TFA) Initial 10 90 10.070 30 10.01-15 90 10

Synthesis of Intermediates Intermediate A3,5-Dichloro-4-aminobenzylamine

To lithium aluminum hydride (0.57 g, 15 mmol) in dry THF (20 mL) wasadded dropwise 3,5-dichloro-4-aminobenzonitrile (1.87 g, 10 mmol) in THF(30 mL). The mixture was stirred at rt for 2 h. Then, sodium sulfatedecahydrate (4.83 g, 15 mmol) was added and the mixture was stirred for30 min. The solid was filtered off and washed with THF three times. Thesolvent was removed under vacuum and the residue was purified bychromatography with Methanol/DCM (3:7) as the eluant.3,5-Dichloro-4-aminobenzylamine was obtained as an off-white solid (1.5g, 80%). ¹H NMR (300 MHz, CD₃OD): δ 3.65 (s, 2H), 7.2 (s, 2H).

Intermediate B 4-Acetamido-3,5-dichloro-benzylamine

Step B (1): To a solution of 3,5-dichloro-4-amino-benzonitrile (187 mg,1 mmol) in 4 mL of THF at room temperature was added 2.2 mL of 1.0 MNaHMDS in THF. The resulting reaction mixture was stirred at roomtemperature for 30 min, at which time acetyl chloride (3.1 mmol) wasadded. DCM (100 mL) and water (100 mL) were added to the reactionmixture after being stirred overnight, followed by the addition of 5 mLof a 1.4 N HCl aqueous solution. The layers were separated and theaqueous layer was extracted with DCM (2×100 mL). The extracts werecombined and solvents were evaporated in vacuo. The residue was purifiedby HPLC to give 4-acetamido-3,5-dichloro-benzonitrile. MS (ESI)(M−H)⁺=227.05. ¹H NMR (300 MHz, CDCl₃) δ 7.63 (s, 2 H), 2.21 (s, 3H).

Step B (2): To a solution of 4-acetamido-3,5-dichloro-benzonitrile (114mg, 0.5 mmol) in 5 mL of THF was added lithium aluminum hydride (120 mg,3.2 mmol) in one portion and the resulting mixture was stirred for 3 h.Sodium sulfate decahydrate (1 g, 3.2 mmol) was added in one portion andthe mixture was stirred for 30 min. The solid was filtered off and thefiltrate was concentrated. The residue was purified by HPLC to give4-acetamido-3,5-dichloro-benzylamine (35 mg, 30%). ¹H NMR (500 MHz,CD₃OD): δ 7.46 (s, 2 H), 3.78 (s, 2H), 2.18 (s, 3 H).

Intermediate C N-Boc-S-methylisothiourea

To a rapidly stirred suspension of S-Methylisothiourea hemisulfate (60.8g, 0.437 mol) in CH₂Cl₂ (600 mL) was added 2N NaOH (300 mL, 0.6 mol).This mixture was cooled to 0° C. on an ice bath, and a solution ofdi-tert-butyl dicarbonate (43.2 g, 0.198 mol) was added dropwise over 6h. Upon completion of the addition, the mixture was stirred anadditional 20 min, diluted with 1 L of CH₂Cl₂ and the phases wereseparated. The organic portion was washed with water (2×500 ml) anddried over Na₂SO₄. Filtration and concentration provided the desiredN-Boc-S-methylisothiourea as a white solid (35.5 g, 0.187 mol, 94% yieldbased on Boc₂O).

Intermediate D (S)-tert-Butylmethylthio-N-(1-phenylpyrrolidine-5-carbonyl)carbonoimidoylcarbamate

Step D (1): To a sealed tube flushed with nitrogen was added L-proline(3.684 g, 32.0 mmol), potassium carbonate (6.634 g, 48.0 mmol), copper(I) iodide (609 mg, 3.2 mmol), iodobenzene (3.58 mL, 32.0 mmol) andN,N-dimethylacetamide. The mixture was heated at 90° C. for 48 hours,then cooled to room temperature. Water was added, and the pH adjusted to<3 with concentrated HCl. The aqueous phase was extracted 4 times withethyl acetate. The combined organic layers were washed with brine, driedover magnesium sulfate, filtered and concentrated in vacuo. Silica gelchromatography (0 to 100% EtOAc/hexane gradient) affordedN-phenylproline which was used directly in the next step. LC-MS(M+H)⁺=192. LC-MS RT (column 1)=2.230/4.00. ¹H NMR (500 MHz, CDCl₃): δ10.574 (br s, 1H), 7.29-7.25 (m, 2H), 6.785 (t, J=7.5 Hz, 1H), 6.613 (d,J=8.0 Hz, 2H), 4.25 (dd, J=2.4, 8.8 Hz, 1H), 3.60 (dt, J=2.8, 8.3 Hz,1H), 3.36 (m, 1H), 2.38-2.01 (m, 4H).

Step D (2): To a solution of the product from step D(1) (151 mg, 791umol) and Intermediate C (158 mg, 834 umol) in DMF (7.3 mL) was addedHATU (381 mg, 1.0 mmol), then NMM (321 uL, 2.92 mmol). The resultingsolution was stirred at room temperature overnight. The solution wasthen diluted with ethyl acetate and washed with 1 M HCl, water, andbrine. The organic layer was then dried with magnesium sulfate, filteredand concentrated in vacuo. Silica gel chromatography (0 to 50%EtOAc/hexane gradient) afforded (S)-tert-butylmethylthio-N-(1-phenylpyrrolidine-5-carbonyl)carbonoimidoylcarbamate(231.2 mg, 81%). LC-MS (M+H)⁺=364. LC-MS RT (column 1)=3.020/4.00. ¹HNMR (500 MHz, CD₃OD): δ 7.21 (t, J=7.9 Hz, 2H), 6.75 (t, J=7.3 Hz, 1H),6.60 (d, J=7.9 Hz, 2H), 4.08 (dd, J=2.1, 9.5 Hz, 1H), 3.81 (t, J=6.9 Hz,1H), 3.32-3.25 (m, 1H), 2.44-2.33 (m, 1H), 2.32 (s, 3H), 2.22-1.98 (m,3H), 1.34 (s, 9H). ¹³C NMR (500 MHz, CD₃OD): 174.41, 168.05, 160.33,147.15, 129.30, 118.25, 112.99, 81.15, 78.47, 64.43, 49.34, 31.43,27.34, 27.15, 24.10, 13.36.

Intermediate E (S)-tert-ButylN-(1-(4-methoxyphenyl)pyrrolidine-5-carbonyl)(methylthio)carbonoimidoylcarbamate

Step E (1): In a 3-necked flask, L-proline (17.25 g, 150 mmol),p-bromoanisole (19.4 mL, 155 mmol), potassium carbonate (21.4 g, 155mmol), and copper(II) oxide (1.00 g, 12.6 mmol) were combined. DMF (25mL) was added, the vessel flushed with argon, and the reaction heated ina 170° C. oil bath overnight. The reaction was cooled to roomtemperature, water was added, and the mixture filtered through Celite.The aqueous filtrate was extracted three times with methylene chloride(discarded). The aqueous phase was then adjusted to pH 2.5 withconcentrated HCl. The desired material fell to the bottom of thesepartory funnel as a dense brown oil. The brown oil turned to solidunder a stream of nitrogen, and was used without further purification inthe subsequent reaction. LC-MS (M+H)⁺=222. LC-MS RT (column3)=1.563/5.00.

Step E (2): The product from step E(1) above was subjected to thereaction conditions of step D(2) to afford Intermediate E in 60% yield.LC-MS (M+H)⁺=394. LC-MS RT (column 2)=4.010/5.00. ¹H NMR (500 MHz,CDCl₃): δ 6.79 (d, J=9.2 Hz, 2H), 6.52 (d, J=8.5 Hz, 2H), 3.99 (d, J=7.9Hz, 1H), 3.81-3.75 (m, 1H), 3.70 (s, 3H), 3.21 (q, J=8.3 Hz, 1H),2.37-2.26 (m, 4H), 2.23-2.15 (m, 1H), 2.08-1.98 (m, 2H), 1.33 (s, 9H).

Intermediate FN-(4-(Aminomethyl)-2,6-dichlorophenyl)-2-(dimethylamino)acetamide

Step F (1): To a solution of the compound of Intermediate A (9 g, 0.047mol) in dry THF under N₂, diisopropyl ethylamine (8.2 ml, 0.047 mol) wasadded. The reaction mixture was cooled to 0° C. and then Boc anhydride(10.26 g, 0.047 mol) dissolved in dry THF (25 mL) was added drop wisemaintaining the reaction temperature at 0° C. After the addition wasover, the reaction mixture was allowed to reach room temperature andstirred for about 2 hrs. After ensuring the absence of the startingmaterial, THF was removed under the vacuum and the resulting solid wasdissolved in ethyl acetate. The organic layer was washed with water (25mL), brine (25 mL), dried over anhydrous sodium sulfate and concentratedunder vacuum to afford the product as pale yellow colored solid. Theproduct was further purified by recrystallization (9.9 g, 72.2%) usingpet ether/ether.

Step F (2): To a suspension of NaH (1.37 g, 0.034 mol) in dry DMF (5 mL)under N₂, the compound of step F (1) (10 g, 0.034 mol) was added at 0°C. The reaction mixture was stirred for about 30 min. at roomtemperature. The reaction mixture was again cooled to 0° C. andbromoacetyl bromide (7.62 g, 0.037 mol) was added and allowed to stirovernight at room temperature. The reaction mixture was poured on tocrushed ice and the precipitate was extracted with ethyl acetate (3×50ml). The organic layer was washed with water (25 ml), brine (25 ml),dried over anhydrous sodium sulfate and concentrated under vacuum. Theresulting product was purified by column chromatography using CHCl₃:MeOH(9:1) as eluent to afford the product as brown colored solid (6 g,42.5%)

Step F (3): To a solution of the compound of step F (2) (6 g 0.014 mol)in dry DMF (60 ml) under N₂, anhydrous K₂CO₃ (6.03 g, 0.043 mol) wasadded with stirring. The reaction mixture was cooled to 0° C. andN,N-dimethylamine hydrochloride (2.37 g, 0.029 mol) was added at once.The reaction mixture was allowed stir for overnight at room temperature.The reaction mixture was poured into water and extracted with ethylacetate (2×150 ml). The organic layer was washed with water (25 ml),brine (25 ml), dried over anhydrous sodium sulfate and concentratedunder vacuum to remove the volatiles. The resulting crude product waspurified by column chromatography using CHCl₃: MeOH (9:1) as eluent toafford the product as an off-white solid (4 g, 72.9%)

Step F (4): To a solution of the compound of step F (3) (12 g) in drydioxane (50 ml), HCl in dioxane (100 ml) was added while stirring. Thereaction mixture was heated to 50° C. overnight. After ensuring theabsence of the starting material, the reaction mixture was concentratedunder vacuum to remove the dioxane and the obtained solid was washedwith pet ether/ether mixture to provide the pure product as a whitesolid in its HCl salt form (9 g, 90.4%) ¹H NMR (400 MHz, (CD₃)₂SO): δ11.05 (s, 1 H), 10.2 (br s, 1 H), 8.63 (br s, 2 H), 7.75 (s, 2 H), 4.24(s, 2 H), 4.05 (s, 2 H), 2.87 (s, 6 H).

Intermediate GN-(4-(Aminomethyl)-2-chloro-6-methylphenyl)-2-(dimethylamino)acetamide

The title compound was prepared as outlined in the scheme following thegeneral procedures for the preparation of Intermediate F.

LC/MS Method A: Column: XTERRA 4.6×30 mm S5; Flow Rate: 4 mL/min.;Solvent A: 10% MeOH—90% water—0.1% TFA; Solvent B: 90% MeOH—10%water—0.1% TFA; Gradient: % B 0-100; Gradient Time: 3 min.

Step G (1): 4-(N-Boc-Aminomethyl)-2-chloro-6-methylbenzenamine

¹H NMR (500 MHz, CDCl₃) δ 7.06 (s, 1 H), 6.88 (s, 1 H), 4.15 (s, 2 H),2.18 (s, 3 H), 1.45 (s, 9 H); LC/MS RT (Method A)=2.841 min.; (M+H)⁺=271(base peak 215).

Step G (2):N-(4-(N-Boc-Aminomethyl)-2-chloro-6-methylphenyl)-2-bromoacetamide

¹H NMR (500 MHz, CDCl₃) δ 7.87 (s, 1 H), 7.21 (s, 1 H), 7.07 (s, 1 H),4.85 (s, 1 H), 4.25 (d, J=5.5 Hz, 2 H), 4.06 (s, 2 H), 2.25 (s, 3 H),1.46 (s, 9 H); LC/MS RT (Method A)=2.866 min.; (M+H)⁺=none (base peak369.04).

Step G (3):N-(4-(N-Boc-Aminomethyl)-2-chloro-6-methylphenyl)-2-(dimethylamino)acetamide

¹H NMR (500 MHz, CDCl₃) δ 8.82 (s, 1 H), 7.18 (s, 1 H), 7.04 (s, 1 H),4.91 (s, 1 H), 4.22 (d, J=5.5 Hz, 2 H), 3.12 (s, 2 H), 2.43 (s, 6 H),2.24 (s, 3 H), 1.44 (s, 9 H); LC/MS RT (Method A)=2.330 min.;(M+H)⁺=356.2.

Step G (4):N-(4-(Aminomethyl)-2-chloro-6-methylphenyl)-2-(dimethylamino)-acetamide

The title compound was prepared from4-(aminomethyl)-2-chloro-6-methylbenzenamine following the generalprocedures as described for the preparation of Intermediate F. ¹H NMR(500 MHz, CD₃OD) δ 7.48 (s, 1 H), 7.35 (s, 1 H), 4.28 (s, 2 H), 4.10 (s,2 H), 3.01 (s, 6 H), 2.31 (s, 3 H); LC/MS (Method A) RT=0.193 min.;(M+H)⁺=256.

Intermediate H (S)-tert-ButylN-(1-pyrrolidine-2-carbonyl-4-trans-methoxy)(methylthio)carbonoimidoylcarbamate

Step H (1): To a suspension of trans-L-4-hydroxy proline (28.17 g, 215mmol) in 2:1 THF:H₂O (700 mL) was added 2.5 M NaOH (118 mL), then Boc₂O(67.1 mL, 292 mmol). The reaction was stirred overnight at roomtemperature. The THF was then removed in vacuo, and 10% KHSO₄ solution(423 ml) was added. The mixture was extracted three times with ethylacetate. The combined organic layers were partitioned with brine, driedover MgSO₄, and concentrated to afford trans-L-N-Boc-4-hydroxy proline(44.41 g, 89% yield). ¹H NMR (500 MHz, DMSO-d₆) δ ppm 12.49 (s, 1 H),4.86-5.23 (m, 1 H), 4.20-4.30 (m, 1 H), 4.07-4.16 (m, 1 H), 3.31-3.46(m, 1 H), 3.19-3.29 (m, 1H), 2.03-2.18 (m, 1 H), 1.81-1.95 (m, 1 H),1.39 (s, 3 H), 1.34 (s, 6 H).

Step H (2): The material from step H (1) (9.02 g, 39.1 mmol) in DMF(66.7 mL) was slowly added to a 0° C. suspension of NaH (3.90 g, 97.5mmol as a 60% dispersion in oil) in DMF (83.5 mL). After the additionwas complete (˜45 minutes), the reaction was stirred an additional 15min at 0° C., then for 30 min at room temperature. The reaction wasagain cooled to 0° C., and iodomethane (2.43 ml, 39.0 mmol) was added.The reaction was allowed to come to room temperature, and stirred atthis temperature for 2 h. The reaction was carefully quenched with 1 MHCl (100 mL). The mixture was extracted three times with EtOAc. Thecombined organics were washed with water, then brine, and dried overMgSO₄. The extracts were concentrated in vacuo then subjected to silicagel chromatography (0 to 20% MeOH/CHCl₃ gradient) to afford the desiredtrans-L-N-Boc-4-methoxy proline (6.19 g, 65% yield).

Step H (3): To a solution of the product from step H (2) (6.19 g, 25.5mmol) in dichloromethane (112 mL) was added TFA (12.6 mL). After 3 h,the volatiles were removed in vacuo. The residue was taken up in pH 7buffer, then lyophilized to produce trans-L-4-methoxy proline, which wasused directly in the subsequent experiment.

Step H (4): trans-L-4-methoxy proline (step H (3)) was reacted in themanner described in step E (1) but using bromobenzene to producetrans-L-N-phenyl-4-methoxy proline (2.667 g, 48% yield). LC-MS(M+H)⁺=222. LC-MS RT (column 1)=1.790/5.00.

Step H (5): trans-L-N-phenyl-4-methoxy proline (step H (4)) (2.667 g,12.07 mmol) was reacted as described in step D (2) to produce thedesired (S)-tert-butylN-(1-pyrrolidine-2-carbonyl-4-trans-methoxy)(methylthio)-carbonoimidoylcarbamate(3.99 g, 84% yield). LC-MS (M+H)⁺=294. LC-MS RT=3.301/5.00. ¹H NMR (500MHz, CDCl₃) δ ppm 12.56 (s, 0.7 H), 12.19 (s, 0.3 H), 7.12-7.23 (m, 2H), 6.65-6.83 (m, 1 H), 6.48-6.61 (m, 2 H), 4.12-4.55 (m, 2 H),3.97-4.06 (br s, 0.7 H), 3.80 (br s, 0.3 H), 3.37-3.48 (m, 1 H),3.31-3.36 (s, 3 H), 2.25-2.55 (m, 5 H), 1.28-1.55 (m, 9 H).

Intermediate I cis-tert-ButylN-(3-phenyl-3-azabicyclo[3.1.0]hexane-2-carbonyl)(methylthio)carbonoimidoylcarbamate

Step I (1): In a sealed tube, cis-3-azabicyclo[3.1.0]hexane-2-carboxylicacid (547 mg, 4.3 mmol), iodobenzene (496 μL, 4.43 mmol), potassiumcarbonate (611 mg, 4.43 mmol), and copper(II) oxide (41 mg, 0.516 mmol)were combined. DMF (1.1 mL) was added, the vessel flushed with nitrogen,and the reaction heated in a 170° C. oil bath overnight. The reactionwas cooled to room temperature, water was added, and the mixturefiltered through Celite. The aqueous filtrate was extracted three timeswith methylene chloride (discarded). The aqueous phase was then adjustedto pH 2.5 with concentrated HCl and extracted three times with ethylacetate. The combined ethyl acetate extracts were washed with saturatedaqueous sodium bicarbonate, dried (MgSO₄), filtered, and concentrated invacuo to give a brown oil (263 mg, 30%) which was used without furtherpurification in subsequent reaction. LC-MS (M+H)⁺=204. LC-MS RT (column2)=1.688/3.000.

Step I (2): The product from step I (1) above was subjected to thereaction conditions of step D (2) to afford Intermediate I in 42% yield.LC-MS (M+H)⁺=376. LC-MS RT (column 2)=2.247/3.000. ¹H NMR (300 MHz,CDCl₃) δ ppm 7.13-7.21 (m, 2 H), 6.79 (t, J=7.50 Hz, 1 H), 6.59 (d,J=8.05 Hz, 2 H), 4.22 (br s, 1 H), 3.96 (d, J=8.78 Hz, 1H), 3.38 (dd,J=8.78, 4.76 Hz, 1 H), 2.36 (s, 3 H), 2.04-2.12 (m, 1 H), 1.80 (ddd,J=11.62, 7.78, 4.39 Hz, 1 H), 1.35 (s, 9 H), 1.25 (t, J=7.14 Hz, 1 H),0.85 (br s, 1 H), 0.69-0.78 (m, 1 H)

EXAMPLE 1(S)—N-((4-Amino-3,5-dichlorobenzylamino)(amino)methylene)-1-phenylpyrrolidine-2-carboxamide

Step 1 (A): (S)-tert-ButylN-(4-amino-3,5-dichlorobenzyl)-N′-(1-phenylpyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a suspension of Intermediate D (150 mg, 413 umol) and Intermediate A(157.8 mg, 826 umol) in CH₂Cl₂ (6.4 mL) was added DIPEA (143 uL, 821umol). The mixture was stirred at rt overnight. Solvents were removed invacuo. The residue was loaded onto a silica gel chromatography column,and eluted with a 0 to 75% EtOAc/hexane gradient to afford the pureproduct (181.8 mg, 87% yield). LC-MS (M+H)⁺=506. LC-MS RT (column1)=2.806/4.00. ¹H NMR (500 MHz, CD₃OD, rotamers seen): 67.23-7.15 (m,3.3H), 7.14-7.08 (m, 0.7H), 6.73 (t, J=7.3 Hz, 0.7H), 6.63-6.55 (m,1.7H), 6.49 (d, J=7.9 Hz, 0.6H), 4.42-4.30 (m, 2H), 4.19-4.14 (dd,J=2.4, 8.9 Hz, 0.3 H), 4.11-4.06 (dd, J=2.4, 9.5 Hz, 0.7 Hz), 3.86-3.80(m, 0.7H), 3.56-3.48 (m, 0.3 H), 3.31-3.24 (m, 1H), 2.41-2.28 (m, 1 H),2.22-2.14 (m, 1 H), 2.11-2.03 (m, 2 H), 1.47 (s, 2.7H), 1.34 (s, 6.3 H).¹³C NMR (500 MHz, CD₃OD, rotamers seen): δ 177.56, 163.08, 155.35,147.07, 140.60, 129.23, 128.94, 127.74, 127.60, 127.53, 119.27, 118.03,115.86, 112.90, 112.11, 83.69, 79.56, 78.45, 65.96, 64.55, 49.16, 43.28,43.15, 31.56, 31.47, 27.44, 27.21, 24.01, 23.91.

Step 1 (B):(S)—N-((4-Amino-3,5-dichlorobenzylamino)(amino)-methylene)-1-phenylpyrrolidine-2-carboxamide

To a solution of the product of step (A) (81.3 mg, 161 umol) in CH₂Cl₂(3.6 mL) was added TFA (360 uL). The mixture was stirred at rt for 2.5h, and then the mixture was concentrated to give the title compoundafter prep HPLC. LC-MS (M+H)⁺=406. LC-MS RT (column 1)=2.035/4.00. ¹HNMR (500 MHz, CD₃OD): δ 7.34-7.18 (m, 4H), 6.83 (t, J=7.3 Hz, 1H), 6.67(d, J=7.9 Hz, 2H), 4.34 (s, 2H), 4.17 (dd, J=2.8, 9.8 Hz, 1H), 3.76 (t,J=7.0 Hz, 1H), 3.28-3.21 (m, 1H), 2.52-2.37 (m, 1H), 2.32-2.18 (m, 1H),2.17-1.98 (m, 2H). ¹³C NMR (500 MHz, CD₃OD): δ 177.99, 153.59, 147.59,141.62, 129.42, 127.68, 123.76, 119.34, 118.84, 113.35, 93.76, 64.88,49.84, 48.83, 44.10, 31.55, 24.02.

EXAMPLE 2(S)—N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)methylene)-1-phenylpyrrolidine-2-carboxamide

Step 2 (A): (S)-tert-ButylN-(4-acetamido-3,5-dichlorobenzyl)-N′-(1-phenylpyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a suspension of Intermediate D (78.2 mg, 215 umol) and Intermediate B(100.3 mg, 431 umol) in CH₂Cl₂ (3.4 mL) was added DIPEA (74.2 uL, 426umol). The mixture was stirred at rt overnight. Solvents were removed invacuo. The residue was loaded onto a silica gel chromatography column,and eluted with a 0% to 75% EtOAc/hexane gradient to afford the pureproduct (94 mg, 76% yield). LC-MS (M+H)⁺=548. LC-MS RT (column2)=2.062/3.00.

Step 2 (B):(S)—N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)-methylene)-1-phenylpyrrolidine-2-carboxamide

To a solution of the product of step (A) (89 mg, 162 umol) in CH₂Cl₂(3.6 mL) was added TFA (360 uL). The mixture was stirred at rt for 2.5h, and then the mixture was concentrated to give the title compoundafter prep HPLC. LC-MS (M+H)⁺=448. LC-MS RT (column 1)=2.313/4.00. ¹HNMR (500 MHz, CD₃OD): δ 7.47 (s, 2H), 7.26 (d, J=7.9 Hz, 2H), 6.82 (t,J=7.3 Hz, 1H), 6.68 (d, J=8.2 Hz, 2H), 4.53 (s, 2H), 4.20 (dd, J=2.4,9.8 Hz, 1H), 3.78 (t, J=6.4 Hz, 1H), 3.31-3.25 (m, 1H), 2.52-2.39 (m,1H), 2.31-2.24 (m, 1H), 2.19 (s, 3H), 2.15-2.04 (m, 2H). ¹³H NMR (500MHz, CD₃OD): δ 217.97, 178.02, 171.26, 154.20, 147.55, 136.94, 134.95,129.42, 127.48, 118.70, 113.30, 64.72, 49.79, 43.80, 31.54, 24.01,21.33.

EXAMPLE 3(S)—N-((3,5-Dichloro-4-(2-(dimethylamino)-acetamido)-benzylamino)(amino)methylene)-1-phenylpyrrolidine-2-carboxamide

Step 3 (A): (S)-tert-ButylN-(3,5-dichloro-4-(2-(dimethylamino)-acetamido)benzyl)-N′-(1-phenylpyrrolidine-5-carbonyl)-carbamimidoylcarbamate

To a solution of Example 1, step (A) (55.6 mg, 110 umol) in CH₂Cl₂ (1.5mL) under nitrogen was added triethylamine (17.5 uL, 126 umol), followedby bromoacetyl bromide (10.6 uL, 121 umol). After 1 h, the reaction was˜30% complete by TLC. Another aliquot of bromoacetyl bromide 10.6 uL,121 umol) was added. After another 1 h, TLC revealed no startingmaterial. Solvents were removed in vacuo. The crude material was usedwithout further purification in the next step. LC-MS (M+H)⁺=626. LC-MSRT (column 1)=2.705/4.00.

Step 3 (B): To a solution of the product of step (A) (110 umol) inCH₂Cl₂ (1.5 mL) was added 2M dimethylamine in THF (1.6 mL, 3.2 mmol).After 2 h, a precipitate had formed, and the reaction was complete byLC/MS. Solvents were removed in vacuo. The residue was loaded onto asilica gel chromatography column, and eluted with a 0 to 100%EtOAc/hexane gradient to afford the pure product (53.3 mg, 82% yield).LC-MS (M+H)⁺=591. LC-MS RT (column 1)=2.372/4.00.

Step 3 (C):(S)—N-((3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(amino)methylene)-1-phenylpyrrolidine-2-carboxamide

To a solution of the product of step (B) (82.6 mg, 140 umol) in CH₂Cl₂(3.1 mL) was added TFA (310 uL). The mixture was stirred at rt for 2.5h, and then the mixture was concentrated to give the title compoundafter prep HPLC. LC-MS (M+H)⁺=491. LC-MS RT (column 1)=1.445/4.00. ¹HNMR (500 MHz, CD₃OD): δ 7.53 (s, 2H), 7.26 (t, J=7.9 Hz, 2H), 6.81 (t,J=7.3 Hz, 1H), 6.67 (d, J=7.9 Hz, 2H), 4.57 (s, 2H), 4.30 (s, 2H), 4.21(dd, J=2.7, 9.8 Hz, 1H), 3.82 3.75 (m, 1H), 3.32-3.25 (m, 1H), 3.06-2.99(s, 6H), 2.51-2.40 (m, 1H), 2.31-2.24 (m, 1H), 2.15-2.04 (m, 2H). ¹³CNMR (500 MHz, CD₃OD): δ 178.06, 163.85, 154.29, 147.54, 137.75, 134.65,131.37, 129.39, 127.64, 118.64, 113.28, 64.68, 58.09, 49.77, 43.77,43.45, 31.52, 23.98.

EXAMPLE 4(S)—N-((4-Amino-3,5-dichlorobenzylamino)(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

Step 4 (A): (S)-tert-ButylN-(4-amino-3,5-dichlorobenzyl)-N′-(1-(4-methoxyphenyl)pyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a suspension of Intermediate E (417.5 mg, 1.06 mmol) and IntermediateA (404.7 mg, 2.12 mmol) in CH₂Cl₂ (16.5 mL) was added DIPEA (366 uL,2.10 mmol). The mixture was stirred at rt overnight. Solvents wereremoved in vacuo. The residue was loaded onto a silica gelchromatography column, and eluted with a 0 to 75% EtOAc/hexane gradientto afford the pure product (510.8 mg, 90% yield). LC-MS (M+H)⁺=536.LC-MS RT (column 2)=2.083/3.00. ¹H NMR (500 MHz, CDCl₃, major rotamer):δ 7.14 (s, 2H), 6.82 (d, J=9.2 Hz, 2H), 6.53 (d, J=8.9 Hz, 2H),4.45-4.42 (m, 2H), 4.01 (dd, J=2.3, 9.6 Hz, 1H), 3.86-3.80 (m, 1H),3.74-3.71 (s, 3H), 3.28-3.20 (m, 1H), 2.39-2.26 (m, 1H), 2.22-2.04 (m,3H), 1.37 (s, 9H).

Step 4 (B):(S)—N-((4-Amino-3,5-dichlorobenzylamino)-(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

To a solution of the product of step (A) (160.6 mg, 299.7 umol) inCH₂Cl₂ (6.6 mL) was added TFA (660 uL). The mixture was stirred at rtfor 2.5 h, and then the mixture was concentrated to give the titlecompound after prep HPLC. LC-MS (M+H)⁺=436. LC-MS RT (column1)=2.425/5.00. ¹H NMR (500 MHz, CD₃OD): δ 7.24 (s, 2H), 6.87 (d, J=8.9Hz, 2H), 6.62 (d, J=8.9 Hz, 2H), 4.34 (s, 2H), 4.06 (dd, J=2.8, 9.8 Hz,1H), 3.78-3.71 (m, 4H), 3.24-3.14 (m, 1H), 2.48-2.36 (m, 1H), 2.27-2.19(m, 1H), 2.13-1.98 (m, 2H). ¹³C NMR (500 MHz, CD₃OD): δ 178.35, 153.46,142.01, 141.56, 127.69, 123.87, 119.34, 115.05, 114.37, 65.31, 55.15,50.45, 44.05, 31.62, 24.15.

EXAMPLE 5(S)—N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

Step 5 (A): (S)-tert-ButylN-(4-acetamido-3,5-dichlorobenzyl)-N′-(1-(4-methoxyphenyl)pyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a suspension of Intermediate E (146.8 mg, 373.5 umol) andIntermediate B (174.0 mg, 747 umol) in CH₂Cl₂ (5.8 mL) was added DIPEA(128.7 uL, 739 umol). The mixture was stirred at rt overnight. Solventswere removed in vacuo. The residue was loaded onto a silica gelchromatography column, and eluted with a 0 to 75% EtOAc/hexane gradientto afford the pure product (168.1 mg, 78% yield) plus some recoveredstarting material. LC-MS (M+H)⁺=578. LC-MS RT (column 1)=2.425/4.00. ¹HNMR (500 MHz, CD₃OD, rotamers seen): δ 7.33-7.21 (m, 2H), 6.81 (d, J=8.9Hz, 1.4H), 6.74 (d, J=9.2 Hz, 0.6H), 6.53 (d, J=9.2 Hz, 1.4H), 6.47-6.36(m, 0.6H), 4.62-4.38 (m, 2H), 4.10 (q, J=7.0 Hz, 1.3H), 4.02 (dd, J=2.1,9.5 Hz, 0.7H), 3.86-3.79 (m, 0.8H), 3.73-3.70 (m, 3.2H), 3.32-3.19 (m,1H), 2.39-2.28 (m, 0.7H), 2.27-2.03 (m, 5.3H), 1.44 (s, 2.8H), 1.35 (s,6.2H).

Step 5 (B):(S)—N-((4-acetamido-3,5-dichlorobenzylamino)(amino)-methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

To a solution of the product of step (A) (168 mg, 291 umol) in CH₂Cl₂(6.4 mL) was added TFA (640 uL). The mixture was stirred at rt for 2.5h, and then the mixture was concentrated in vacuo. Prep HPLC, followedby flash chromatography (silica gel, MeOH/chloroform gradient) affordedthe title compound. LC-MS (M+H)⁺=478. LC-MS RT (column 2)=2.115/5.00. ¹HNMR (500 MHz, CD₃OD): δ 7.39 (s, 2H), 6.77 (d, J=6.7 Hz, 2H), 6.49 (brs, 2H), 4.42 (s, 2H), 4.00 (d, J=6.4 Hz, 1H), 3.71 (s, 3H), 3.62-3.44(m, 1H), 3.25 (q, J=7.7 Hz, 1H), 2.37-2.23 (m, 1H), 2.19 (s, 3H),2.14-1.90 (m, 3H).

EXAMPLE 6(S)—N-((3,5-Dichloro-4-(2-(dimethylamino)acetamido)benzylamino)-(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

Step 6 (A): To a solution of Example 1, step (A) (200.0 mg, 373.2 umol)in CH₂Cl₂ (4.9 mL) under nitrogen was added triethylamine (59.5 uL, 429umol), followed by bromoacetyl bromide (35.9 uL, 411 umol). After 1 h,the reaction was ˜30% complete by TLC. Another aliquot of bromoacetylbromide 35.9 uL, 411 umol) was added. After an additional hour, thereaction was ˜80% complete by TLC. Another aliquot of bromoacetylbromide 18 uL, 205 umol) was added After another 1 h, TLC revealed nostarting material. Solvents were removed in vacuo. The crude materialwas used without further purification in the next step.

Step 6 (B): (S)-tert-ButylN-(3,5-dichloro-4-(2-(dimethylamino)-acetamido)benzyl)-N′-(1-(4-methoxyphenyl)pyrrolidine-5-carbonyl)carbamimidoylcarbamate

To a solution of the product from step (A) (373 umol) in CH₂Cl₂ (4.9 mL)was added 2M dimethylamine in THF (4.9 mL, 9.8 mmol). After stirringovernight, a precipitate had formed, and the reaction was complete byLC/MS. Solvents were removed in vacuo. The residue was loaded onto asilica gel chromatography column, and eluted with a 0 to 100%EtOAc/hexane gradient to afford the pure product (131.9 mg, 57% yield).LC-MS (M+H)⁺=621. LC-MS RT (column 1)=2.758/5.00. ¹H NMR (500 MHz,CD₃OD, rotamers seen): δ 7.45 (s, 1.5H), 7.39 (s, 0.5H), 6.83 (d, J=8.9Hz, 1.5H), 6.74 (d, J=8.9 Hz, 0.5H), 6.74 (d, J=8.9 Hz, 1.5H), 6.41 (d,J=8.9 Hz, 0.5H), 4.61-4.45 (m, 2H), 4.26-4.20 (dd, J=3.5, 5.7 Hz, 0.3H),4.15-4.00 (m, 1.7H), 3.87-3.79 (m, 0.7H), 3.74-3.68 (m, 3H), 3.49-3.41(m, 0.3H), 3.29-3.22 (m, 1H), 3.21-3.18 (s, 2H), 2.49-2.41 (m, 6H),2.41-2.34 (m, 0.6H), 2.24-2.16 (m, 0.7H), 2.12-2.04 (m, 1.7H), 1.51 (s,2H), 1.34 (s, 7H).

Step 6 (C):(S)—N-((3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(amino)methylene)-1-(4-methoxyphenyl)pyrrolidine-2-carboxamide

To a solution of the product of step (B) (131.9 mg, 212.4 umol) inCH₂Cl₂ (4.7 mL) was added TFA (470 uL). The mixture was stirred at rtfor 3 h, and then the mixture was concentrated in vacuo. The residue wasdissolved in CH₂Cl₂, and partitioned with saturated NaHCO₃. The organicphase was dried over magnesium sulfate, filtered, and concentrated invacuo. Silica gel chromatography (0 to 25% MeOH/chloroform gradient)afforded the title compound. LC-MS (M+H)⁺=521. LC-MS RT (column2)=1.622/5.00. ¹H NMR (500 MHz, CD₃OD): δ 7.35 (s, 2H), 6.75 (br s, 2H),6.45 (br s, 2H), 4.39 (s, 2H), 4.06-3.90 (m, 1H), 3.70 (s, 3H),3.62-3.41 (m, 1H), 3.28-3.14 (m, 3H), 2.45 (s, 6H), 2.36-2.20 (br s,1H), 2.16-1.86 (m, 3H). ¹³C NMR (500 MHz, CD₃OD): δ 171.15, 142.93,127.16, 114.84, 112.90, 78.45, 66.33, 62.56, 55.40, 45.20, 43.13, 31.90,24.09.

EXAMPLES 7-30

The compounds of Examples 7-30 were prepared in a manner similar to thatdescribed for the synthesis of Example 3 (Scheme 3) but using theappropriate amine. The examples were purified by HPLC under standardconditions to provide the final products.

Ex- MS HPLC Ret. ample R₁₀R₁₁N— MW Observed Time (min) 7cyclopropylamino 483.0 483.2 3.60 8 cyclopentylamino 511.1 511.2 3.89 94-methylcyclohexylamino 539.1 539.2 4.38 10 N-methylcyclohexylamino539.1 539.2 4.15 11 azetidino 483.0 483.1 3.62 12 pyrrolidino 497.0497.1 3.64 13 homopiperazino 526.1 526.2 3.39 14 morpholino 513.0 513.23.80 15 thiomorpholino 529.1 529.1 4.15 16 decahydroquinolino 565.2565.2 4.40 17 isopropylamino 485.0 485.2 3.62 18 methylamino 457.0 457.13.47 19 benzylamino 533.1 533.2 4.16 20 4-methylbenzylamino 547.1 547.24.42 21 ethylamino 471.0 471.2 3.53 22 2-(N,N- 514.1 514.2 2.96dimethylamino)ethylamino 23 2-methoxyethylamino 501.0 501.1 3.59 242-hydroxyethylamino 487.0 487.1 3.45 25 phenethylamino 547.1 547.2 4.3726 N-methybenzylamino 547.1 547.2 4.35 27 N-methylbutylamino 513.1 513.24.01 28 N-methylisopropylamino 499.1 499.2 3.64 292-(thiophen-2-yl)ethylamino 553.1 553.2 4.26 30 cyclohexylmethylamino539.1 539.2 4.48

EXAMPLE 31(R)—N-(Amino(3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)methylene)-1-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carboxamide

Step 31 (A): (R)-tert-Butyl1-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carboxylate

A suspension of 4-(trifluoromethyl)phenylboronic acid (190 mg), copper(II) acetate (27 mg) and 4 Å molecular sieves (188 mg) indichloromethane (4 mL) was stirred at room temperature for 5 min, and(R)-tert-butyl pyrrolidine-2-carboxylate (86 mg) in acetonitrile (0.5mL) was added followed by triethylamine (0.07 mL). The reaction vial wasflushed with oxygen, sealed and stirred at 50° C. for 24 h. The reactionmixture was filtered through a pad of Celite, and the filtrate wasevaporated in vacuo. The residue was purified by preparative TLC elutingwith 10% ethyl acetate/90% hexanes to give the title compound as acolorless oil (43 mg). HPLC retention time: 1.99 min (method A). MS(ESI) (M+H)⁺ 316.14. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (1H, d, J=8.4 Hz),6.52 (1H, d, J=8.4 Hz), 4.15 (1H, m), 3.53 (1H, m), 3.40 (1H, m),2.0-2.3 (3H, m), 1.41 (9H, s).

Step 31 (B): (R)-1-(4-(Trifluoromethyl)phenyl)pyrrolidine-2-carboxylicacid

To a solution of (R)-tert-butyl1-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carboxylate (40 mg) indichloromethane (0.10 mL) was added TFA (0.10 mL), and the resultingsolution was stirred at room temperature for 5.5 h. The solvents wereremoved to give the title compound as the TFA salt. This crude productwas used in step (C) without purification. HPLC retention time: 1.88 min(method A). MS (ESI) (M+H)⁺ 260.09.

Step 31 (C): (R)-tert-Butylmethylthio(1-(4-(trifluoromethyl)-phenyl)pyrrolidine-2-carboxamido)methylenecarbamate

A solution of (R)-1-(4-(trifluoromethyl)phenyl)pyrrolidine-2-carboxylicacid from step (B), tert-butyl(4-amino-2,6-dichlorobenzylamino)(amino)-methylenecarbamate (60 mg),Py•BOP (186 mg) and triethylamine (0.15 mL) in dichloromethane (0.20 mL)was stirred at room temperature for 12 h. The crude product was purifiedby preparative TLC eluting with 50% ethyl acetate/50% hexanes to givethe title compound as a colorless oil (24 mg). retention time: 2.31 min(method A). MS (ESI) (M+Na)⁺ 454.12.

Step 31 (D): (R)-tert-Butyl(3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(1-(4-(trifluoromethyl)phenyl)-pyrrolidine-2-carboxamido)-methylenecarbamate

A solution of (R)-tert-butylmethylthio(1-(4-(trifluoromethyl)-phenyl)pyrrolidine-2-carboxamido)methylenecarbamate(6 mg), 3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamine (10 mg)and triethylamine (50 μL) was heated at 56° C. for 12 h. The crudereaction mixture was purified directly by preparative TLC eluting with10% methanol/89% dichloromethane/1% ammonia hydroxide to give the titlecompound as a colorless oil (5 mg). retention time: 2.03 min (method A).MS (ESI) (M+Na)⁺ 659.19.

Step 31 (E):(R,E)-N-(amino(3,5-dichloro-4-(2-(dimethylamino)-acetamido)-benzylamino)methylene)-1-(4-(trifluoromethyl)phenyl)-pyrrolidine-2-carboxamide

To a solution of: (R)-tert-butyl(3,5-dichloro-4-(2-(dimethylamino)-acetamido)benzylamino)(1-(4-(trifluoromethyl)phenyl)-pyrrolidine-2-carboxamido)-methylenecarbamate(4 mg) in dichloromrthane (0.10 mL) was added TFA (0.50 μL), and theresulting mixture was stirred at room temperature for 12 h. The solventswere removed in vacuo to give the title compound as its TFA salt (3 mg).retention time: 1.52 min (method A). MS (ESI) (M+H)⁺ 559.13.

EXAMPLE 32(2S,4R)—N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)-methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

Step 32 (A):(2S,4R)—N-((4-Acetamido-3,5-dichlorobenzylamino)-(tert-butoxycarbonylamino)methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

Intermediate H and Intermediate B were reacted in the manner of Example1, step (A), to produce the title compound (93% yield). LC-MS(M+H)⁺=578. LC-MS RT (column 1)=2.548/4.00. ¹H NMR (500 MHz, CDCl₃) δppm 12.85 (s, 0.4 H), 12.03 (s, 0.6 H), 8.82-8.97 (m, 1 H), 7.76-7.88(m, 1 H), 7.07-7.33 (m, 4 H), 6.72-6.78 (m, 0.4 H), 6.44-6.65 (m, 2.6H), 3.97-4.60 (m, 5.4 H), 3.65-3.73 (m, 0.6 H), 3.26-3.42 (m, 4 H),2.42-2.53 (m, 0.4 H), 2.19-2.36 (m, 1.6 H), 2.17-2.07 (m, 3H), 1.42 (s,5.4 H), 1.34 (s, 3.6 H).

Step 32 (B): The product of step (A) was reacted in the manner ofExample 1, step (B), to produce(2S,4R)—N-((4-acetamido-3,5-dichlorobenzylamino)(amino)-methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide(72% yield). LC-MS (M+H)⁺=478. LC-MS RT (column 1)=2.018/5.00. ¹H NMR(500 MHz, CD₃OD) δ ppm 7.33 (s, 2 H), 7.12 (s, 2 H), 6.46-6.68 (m, 3 H),4.07-4.44 (m, 4 H), 3.61-3.90 (m, 1 H), 3.26-3.42 (m, 4 H), 2.28-2.46(m, 1 H), 2.14-2.24 (m, 4 H).

EXAMPLE 33(2S,4R)—N-(Amino(3,5-dichloro-4-(2-(dimethylamino)acetamido)benzylamino)methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

Step 33 (A):(2S,4R)—N-((tert-Butoxycarbonylamino)(3,5-dichloro-4-(2-(dimethylamino)acetamido)benzylamino)methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

Intermediate G and Intermediate F were reacted in the manner of Example1, step (A) to produce the title compound (78% yield). LC-MS (M+H)⁺=621.LC-MS RT (column 1)=2.293/4.00. ¹H NMR (500 MHz, CDCl₃) δ ppm 12.83 (s,0.4H), 12.05 (s, 0.6 H), 8.81-9.21 (m, 2 H), 7.10-7.32 (m, 4 H),6.45-6.79 (m, 3H), 3.99-4.62 (m, 4.4 H), 3.67-3.74 (m, 0.6 H), 3.28-3.42(m, 4 H), 3.21 (s, 2H), 2.43-2.52 (m, 6.4 H), 2.22-2.35 (s, 1.6 H), 1.43(s, 5.4 H), 1.34 (s, 3.6 H).

Step 33 (B):(2S,4R)—N-(Amino(3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)methylene)-4-methoxy-1-phenylpyrrolidine-2-carboxamide

The product of step (A) was reacted in the manner of Example 1, step(B), to produce the title compound (84% yield). LC-MS (M+H)⁺=521. LC-MSRT (column 1)=1.502/5.00. ¹H NMR (500 MHz, CD₃OD) δ ppm 7.34 (s, 2 H),7.05-7.21 (s, 2 H), 6.42-6.69 (m, 3 H), 4.09-4.44 (m, 4 H), 3.59-3.90(m, 1 H), 3.26-3.43 (m, 4 H), 3.21 (s, 2 H), 2.26-2.50 (m, 7 H),2.13-2.26 (m, 1 H).

EXAMPLE 34cis-N-((4-Acetamido-3,5-dichlorobenzylamino)(amino)methylene)-3-phenyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

Step 34 (A): cis-tert-ButylN-(4-acetamido-3,5-dichlorobenzyl)-N′-(3-phenyl-3-azabicyclo[3.1.0]hexane-2-carbonyl)carbamimidoylcarbamate

The title compound was prepared from Intermediate I and Intermediate Bin the same manner as Example 2, step (A) Silica gel chromatography ofthe crude product eluted with a 0-10% MeOH/CHCl₃ gradient afforded thepure product (80 mg, 87% yield). LC-MS (M+H)⁺=560. LC-MS RT (column2)=2.152/3.000.

Step 34 (B):cis-N-((4-Acetamido-3,5-dichlorobenzylamino)-(amino)methylene)-3-phenyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

To a solution of the product of step (A) (80 mg, 142 umol) in CH₂Cl₂(2.0 mL) was added TFA (200 uL). The mixture was stirred at rt for 2.5h, and then the mixture was washed with saturated aqueous sodiumbicarbonate, dried (MgSO₄), filtered, and concentrated in vacuo. Theresidue was loaded onto a silica gel chromatography column, and elutedwith a 0 to 10% MeOH/CHCl₃ gradient to afford the pure product as awhite solid (45 mg, 69% yield). LC-MS (M+H)⁺=460. LC-MS RT (column2)=1.547/3.000. ¹H NMR (500 MHz, CD₃OD) δ ppm 7.41 (s, 2 H), 7.10 (s, 2H), 6.63 (s, 1 H), 6.48 (s, 2 H), 4.38-4.47 (m, 2H), 4.04 (d, J=2.75 Hz,1 H), 3.75 (d, J=7.02 Hz, 1 H), 3.31 (s, 1 H), 2.16 (s, 3 H), 2.01 (brs, 1 H), 1.69 (br s, 1 H), 0.78 (br s, 1 H), 0.57 (br s, 1 H)

EXAMPLE 35cis-N-((3,5-Dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(amino)methylene)-3-phenyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

Step 35 (A): cis-tert-ButylN-(3,5-dichloro-4-(2-(dimethylamino)acetamido)-benzyl)-N′-(3-phenyl-3-azabicyclo[3.1.0]hexane-2-carbonyl)carbamimidoylcarbamate

The title compound was prepared from Intermediate I and Intermediate Fin the same manner as Example 2, step (A). Silica gel chromatography ofthe crude product eluted with a 0 to 10% MeOH/CHCl₃ gradient affordedthe pure product (76 mg, 77% yield).LC-MS (M+H)⁺=603. LC-MS RT (column2)=1.940/3.000.

Step 35 (B):cis-N-((3,5-Dichloro-4-(2-(dimethylamino)acetamido)-benzylamino)(amino)methylene)-3-phenyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

The title compound was prepared from the product of step (A) in the samemanner as Example 34 to afford the pure product as a white solid (28 mg,44% yield). LC-MS (M+H)⁺=503. LC-MS RT (column 2)=1.338/3.000. ¹H NMR(500 MHz, CD₃OD) δ ppm 7.43 (s, 2 H), 7.10 (t, J=7.17 Hz, 2 H), 6.63 (d,J=6.10 Hz, 1 H), 6.48 (s, 2 H), 4.39-4.48 (m, 2 H), 4.04 (d, J=4.27 Hz,1 H), 3.75 (d, J=8.55 Hz, 1 H), 3.27-3.30 (m, 1 H), 3.20 (s, 2 H),2.40-2.46 (m, 6 H), 2.01 (br s, 1 H), 1.70 (br s, 1 H), 0.79 (br s, 1H), 0.57 (br s, 1 H)

Biological Methods

There are a number of methods by which inhibitors of the BACE enzyme canbe identified experimentally. The enzyme can be obtained from membranesamples from natural tissues or cultured cells or can be expressedrecombinantly in a host cell by well known methods of molecular biology.The whole enzyme or a portion thereof can be expressed, for example, inbacterial, insect or mammalian cells to obtain a catalytically activeenzyme species. The enzymatic activity and/or ligand binding capabilityof the enzyme can be assessed within these membrane samples, or theenzyme can be purified to varying extents. As an illustrative example,the nucleic acid sequence encoding the pro and catalytic domains ofhuman BACE can be appended on the 5′ end with an untranslated and signalsequence from the gene for acetylcholinesterase, and on the 3′ end witha sequence encoding a poly-histidine tag. This cDNA can then beexpressed in Drosophila melanogaster S2 cells in which the signal andpro sequences of the transcribed/translated protein are removed bycellular proteases and the catalytic domain, appended by a C-terminalpoly-histidine tag, is secreted out into the cellular medium. The enzymecan then be purified from the culture medium by nickel affinitychromatography by methods well known to those trained in the art[Mallender, W. et al., (2001) “Characterization of recombinant, solublebeta-secretase from an insect cell expression system.” Mol. Pharmacol.59: 619-626]. Similar strategies for expressing and purifying variousforms of BACE in bacterial, mammalian and other cell types would beknown to one skilled in the art. A preferred method for determining thepotency of a test compound in binding to the BACE enzyme is bymonitoring the displacement of a suitable radioligand.

Radioligand displacement assays with a radiolabeled BACE inhibitor (WO2004 013098, compound 3, where the methoxy group is substituted forC(³H)₃) were carried out using standard methods (Keen, M. (1999) inReceptor Binding Techniques (Walker, J. M. ed) p. 106 Humana Press,Totowa, N.J.). The HEK293-9B.A1 cell line, which overexpresses the BACE1enzyme, was derived from HEK293 cells (Simmons, N. L. (1990) A culturedhuman renal epithelioid cell line responsive to vasoactive intestinalpeptide. Exp. Physiol. 75:309-19.) by RAGE™ (Harrington, J. J. et al.(2001) Creation of genome-wide protein expression libraries using randomactivation of gene expression. Nat. Biotechnol. 19:440-5.; U.S. Pat.Nos. 6,410,266 and 6,361,972). T225 flask cultures of HEK293-9B.A1 weregrown to 80% confluency in DMEM supplemented with 2 mM L-glutamine, 10μg/ml penicillin, 10 μg/ml streptomycin, 3 μg/ml puromycin, 100 nMmethotrexate, and 10% fetal bovine serum (Invitrogen, Carlsbad, Calif.),harvested, and resuspended at 2×10⁸ cells per 10 ml of lysis bufferconsisting of 50 mM HEPES pH 7.0 containing a protease inhibitorcocktail of AEBSF 104 μM, aprotinin 80 nM, leupeptin 2 μM, bestatin 4μM, pepstatin A 1.5 μM, and E-64 1.4 μM (0.1% of protease inhibitorcocktail P8340, Sigma-Aldrich, St. Louis, Mo.) at 4° C. The resuspendedcells were homogenized using a Polytron (Brinkman, Westbury, N.Y.) atsetting 6 for 10 sec., then centrifuged at 48,000×g for 10 min. Theresulting pellet was washed by repeating the resuspension,homogenization and centrifugation steps. The final pellet wasresuspended in buffer at 4° C. to yield a total protein concentration of5 mg/ml, then aliquots were frozen in liquid nitrogen for furtherstorage at −70° C. Immediately before carrying out a binding assay, analiquot of cell homogenate was thawed and diluted to a concentration of100 μg/ml in assay buffer consisting of 50 mM HEPES pH 5.0 and 0.1%CHAPSO. Assays were initiated in polypropylene 96-well plates (Costar,Cambridge, Mass.) by the addition of 200 μl of cell homogenate to 50 μlof assay buffer containing 1 nM radioligand (WO 2004 013098, compound 3,where the methoxy group is substituted for C(³H)₃: 80 Ci/mMol) andvarious concentrations of unlabelled compounds, and incubated for 1.5hr. at 25° C. Separation of bound from free radioligand was byfiltration on GFF glass fiber filters (Innotech BiosystemsInternational, Lansing, Mich.) using an Innotech cell harvester. Filterswere washed three times with 0.3 ml of phosphate buffered saline pH 7.0at 4° C. and assessed for radioactivity using a Wallac 1450 Microbetaliquid scintillation counter (PerkinElmer, Boston, Mass.). Ki values ofcompeting compounds were derived through Cheng-Prussoff correction ofIC50 values calculated using XLfit (IDBS, Guildford, UK).

Abbreviations:

-   AEBSF: 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride-   CHAPSO:    3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-   D-MEM: Dulbecco's modified eagle medium-   HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid-   RAGE™: Random Activation of Gene Expression™

The activity of specific compounds described herein and tested in theabove assay is provided in Table 1.

TABLE 1 Compounds of Example Activity Rating 1 ++ 2 ++ 3 ++ 4 ++ 5 ++ 6++ 31 +++ 32 +++ 33 +++ 34 ++ ^(a)Activity based on IC₅₀ values: +++ =<0.1 μM ++ = 0.1-1.0 μM + = >1.0 μMIn Vitro Assay to Identify β-Secretase Inhibitor Based on the Inhibitionof Aβ Formation from Membrane Preparations.

An isolated membrane fraction which contains functionally activeβ-secretase and β-APP substrates can generate β-secretase cleavageproducts including Aβ (Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.;Lewis, M.; Smith, D. W.; Pak, R. PCT Publication WO 01/0175435;Fechteler, K.; Kostka, M.; Fuchs, M. Patent Application No. DE99-19941039; Shearman, M.; Beher, D. et al., Biochemistry, 2000, 39,8698-8704; Zhang, L. Song, L. et al., Biochemistry 2001, 40, 5049-5055).An isolated membrane fraction can be prepared from human derived celllines such as HeLa and H4 which have been transfected with wild type ormutant forms of β-APP or a human alkaline phosphatase β-APP fusionconstruct, and stably express high levels of β-secretase substrates. Theendogenous β-secretase present in the isolated membranes prepared at0-4° C. cleaves the β-APP substrates when the membranes are shifted from0-4 to 37° C. Detection of the cleavage products including Aβ can bemonitored by standard techniques such as immunoprecipitation (Citron,M.; Diehl, T. S. et al., Proc. Natl. Acad. Sci. USA, 1996, 93,13170-13175), western blot (Klafki, H.-W.; Ambramowski, D. et al., J.Biol. Chem. 1996, 271, 28655-28659), enzyme linked immunosorbent assay(ELISA) as demonstrated by Seubert, P.; Vigo-Pelfrey, C. et al., Nature,1992, 359, 325-327, or by a preferred method using time-resolvedfluorescence of the homogeneous sample containing membranes and Aβ(Roberts, S. B.; Hendrick, J. P.; Vinitsky, A.; Lewis, M.; Smith, D. W.;Pak, R. PCT Publication WO 01/0175435; Shearman, M.; Beher, D. et al.,Biochemistry, 2000, 39, 8698-8704). The Aβ present in a homogeneoussample containing membranes can be detected by time-resolvedfluorescence with two antibodies that recognize different epitopes ofAβ. One of the antibodies recognizes an epitope that is present in Aβbut not present in the precursor fragments; preferably the antibodybinds the carboxyl terminus of Aβ generated by the β-secretase cleavage.The second antibody binds to any other epitope present on Aβ. Forexample, antibodies that bind the N-terminal region (e.g., 26D6-B2-B3®SIBIA Neurosciences, La Jolla, Calif.) or bind the C-terminal end (e.g.,9S3.2® antibody, Biosolutions, Newark, Del.) of the Aβ peptide areknown. The antibodies are labeled with a pair of fluorescent adductsthat transfer fluorescent energy when the adducts are brought in closeproximity as a result of binding to the N- and C-terminal ends orregions of Aβ. A lack of fluorescence is indicative of the absence ofcleavage products, resulting from inhibition of β-secretase. Theisolated membrane assay can be used to identify candidate agents thatinhibit the activity of β-secretase cleavage and Aβ production.

A typical membrane-based assay requires 45 μg membrane protein per wellin a 96- or 384-well format. Membranes in a neutral buffer are combinedwith the test compound and shifted from 0-4 to 37° C. Test agents maytypically consist of synthetic compounds, secondary metabolites frombacterial or fungal fermentation extracts, or extracts from plant ormarine samples. All synthetic agents are initially screened at dosesranging from 10-100 μM or in the case of extracts at sufficient dilutionto minimize cytotoxicity. Incubation of the membranes with the testagent will continue for approximately 90 minutes at which timefluorescence labeled antibodies are added to each well for Aβquantitation. The time-resolved fluorescence detection and quantitationof Aβ is described elsewhere (Roberts, S. B.; Hendrick, J. P.; Vinitsky,A.; Lewis, M.; Smith, D. W.; Pak, R. PCT Publication WO 01/0175435;Shearman, M.; Beher, D. et al., Biochemistry, 2000. 39, 8698-8704).Results are obtained by analysis of the plate in a fluorescence platereader and comparison to the mock treated membranes and samples in whichknown amounts of Aβ were added to construct a standard concentrationcurve. A positive acting compound is one that inhibits the Aβ relativeto the control sample by at least 50% at the initial testedconcentration. Compounds of the present application are consideredactive when tested in the above assay if the IC₅₀ value for the testcompound is less than 50 μM. A preferred IC₅₀ value is less than 1 μM. Amore preferred IC₅₀ value is less than 0.1 μM. If a compound is found tobe active then a dose response experiment is performed to determine thelowest dose of compound necessary to elicit the inhibition of theproduction of Aβ.

In Vivo Assays for the Determination of Aβ Reduction by a β-SecretaseInhibitor.

In vivo assays are available to demonstrate the inhibition ofβ-secretase activity. In these assays, animals, such as mice, thatexpress normal levels of APP, β- and γ-secretase or are engineered toexpress higher levels of APP and hence Aβ can be used to demonstrate theutility of β-secretase inhibitors, as demonstrated with γ-secretaseinhibitors [Dovey, H. et al., (2001), J. Neurochem. 76: 173-181]. Inthese assays, β-secretase inhibitors are administered to animals and Aβlevels in multiple compartments, such as plasma, cerebral spinal fluid,and brain extracts, are monitored for Aβ levels using methods previouslyoutlined. For instance, Tg2576 mice, which overexpress human APP, areadministered β-secretase inhibitors by oral gavage at doses that willcause measurable Aβ lowering, typically less than 100 mg/kg. Three hoursafter dosing plasma, brain, and CSF are collected, frozen in liquidnitrogen, and stored at −80° C. until analysis. For Aβ detection, plasmais diluted 15-fold in PBS with 0.1% Chaps while CSF is diluted 15-foldin 1% Chaps with protease inhibitors (5 μg/ml leupeptin, 30 μg/mlaprotinin, 1 mM phenylmethylsulfonylfluoride, 1 μM pepstatin). Brainsare homogenized in 1% Chaps with protease inhibitors using 24 mlsolution/g brain tissue. Homogenates were then centrifuged at 100,000×gfor 1 hr at 4° C. The resulting supernatants were then diluted 10-foldin 1% Chaps with protease inhibitors. Aβ levels in the plasma, CSF, andbrain lysate can then be measured using time-resolved fluorescence ofthe homogenous sample or one of the other methods previously described.

A β-secretase inhibitor is considered active in one of the above in vivoassays if it reduces Aβ by at least 50% at a dosage of 100 mg/kg.

Dosage and Formulation

The compounds of the present application can be administered orallyusing any pharmaceutically acceptable dosage form known in the art forsuch administration. The active ingredient can be supplied in soliddosage forms such as dry powders, granules, tablets or capsules, or inliquid dosage forms, such as syrups or aqueous suspensions. The activeingredient can be administered alone, but is generally administered witha pharmaceutical carrier. A valuable treatise with respect topharmaceutical dosage forms is Remington's Pharmaceutical Sciences, MackPublishing.

The compounds of the present application can be administered in suchoral dosage forms as tablets, capsules (each of which includes sustainedrelease or timed release formulations), pills, powders, granules,elixirs, tinctures, suspensions, syrups, and emulsions. Likewise, theymay also be administered in intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, all using dosageforms well known to those of ordinary skill in the pharmaceutical arts.An effective but non-toxic amount of the compound desired can beemployed to prevent or treat neurological disorders related to β-amyloidproduction or accumulation, such as Alzheimer's disease and Down'sSyndrome.

The compounds of this application can be administered by any means thatproduces contact of the active agent with the agent's site of action inthe body of a host, such as a human or a mammal. They can beadministered by any conventional means available for use in conjunctionwith pharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

The dosage regimen for the compounds of the present application will, ofcourse, vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the species, age, sex, health, medical condition, andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; the route ofadministration, the renal and hepatic function of the patient, and theeffect desired. An ordinarily skilled physician or veterinarian canreadily determine and prescribe the effective amount of the drugrequired to prevent, counter, or arrest the progress of the condition.

Advantageously, compounds of the present application may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three, or four times daily.

The compounds for the present application can be administered inintranasal form via topical use of suitable intranasal vehicles, or viatransdermal routes, using those forms of transdermal skin patches wellknown to those of ordinary skill in that art. To be administered in theform of a transdermal delivery system, the dosage administration will,of course, be continuous rather than intermittent throughout the dosageregimen.

In the methods of the present application, the compounds hereindescribed in detail can form the active ingredient, and are typicallyadministered in admixture with suitable pharmaceutical diluents,excipients, or carriers (collectively referred to herein as carriermaterials) suitably selected with respect to the intended form ofadministration, that is, oral tablets, capsules, elixirs, syrups and thelike, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as lactose,starch, sucrose, glucose, methyl callulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral drug components can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor β-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like.

The compounds of the present disclosure can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamallar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

Compounds of the present disclosure may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamidephenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent disclosure may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, andcrosslinked or amphipathic block copolymers of hydrogels.

Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivatives, magnesiumstearate, stearic acid, and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration preferably contain a watersoluble salt of the active ingredient, suitable stabilizing agents, andif necessary, buffer substances. Antioxidizing agents such as sodiumbisulfite, sodium sulfite, or ascorbic acid, either alone or combined,are suitable stabilizing agents. Also used are citric acid and its saltsand sodium EDTA. In addition, parenteral solutions can containpreservatives, such as benzalkonium chloride, methyl- or propyl-paraben,and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

1. A compound of Formula (I); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy,C₂₋₆alkenyl, C₁₋₆alkyl optionally substituted with OH, C₃₋₆cycloalkyl,or —NH₂, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl, —(CH₂)_(m)—NHC(═O)Ophenyloptionally substituted with halogen, —(CH₂)_(m)—NHC(═O)R₈ and—NHC(═O)R₉; R₂ and R₃ are each independently hydrogen, methyl orhydroxymethyl; p is 0 or 1; m is 0 or 1; R₄ and R₆ are independentlyhydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃in which said phenyl is optionally substituted with one or more groupsselected from halogen, CN, CF₃ and OCH₃; R₅ and R_(5′) are eachindependently hydrogen, C₁₋₆alkyl, or C₁₋₄alkanol; R₇ is hydrogen,halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ isC₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted witha group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is—C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen,C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy,C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyloptionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyloptionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy;or R₁₀ and R₁₁ together with the nitrogen to which they are attached isazetidine, aziridine, pyrrolidine, piperidine, homopiperidine,homopiperazine, morpholine or thiomorpholine, in which each isoptionally substituted with a group selected from halogen, C₁₋₆alkyl andC₁₋₄alkoxy; or a nontoxic pharmaceutically acceptable salt thereof.
 2. Acompound of Formula (II); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, CN, CF₃, OH, —NH₂, C₃₋₆cycloalkyl, C₁₋₆alkoxy,C₂₋₆alkenyl, C₁₋₆alkyl optionally substituted with OH, C₃₋₆cycloalkyl,or —NH₂, —(CH₂)_(m)—NHC(═O)OC₁₋₆ alkyl, —(CH₂)_(m)—NHC(═O)Ophenyloptionally substituted with halogen, —(CH₂)_(m)—NHC(═O)R₈ and—NHC(═O)R₉; p is 0 or 1; m is 0 or 1; R₄ is hydrogen, C₁₋₆alkyl,C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl or CF₃ in which said phenylis optionally substituted with one or more groups selected from halogen,CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN,OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl or C₃₋₆cycloalkyl inwhich each is optionally substituted with a group selected from halogen,CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen orC₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionally substituted withOH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl,—(CH₂)_(m)C₃₋₆cycloalkyl optionally substituted with phenyl orC₁₋₄alkyl; —(CH₂)_(m)phenyl optionally substituted with halogen,hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together with thenitrogen to which they are attached is azetidine, aziridine,pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine orthiomorpholine, in which each is optionally substituted with a groupselected from halogen, C₁₋₆alkyl and C₁₋₄alkoxy; or a nontoxicpharmaceutically acceptable salt thereof.
 3. A compound of Formula(III); or a stereoisomer thereof

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substitutedwith cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉;R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl orCF₃ in which said phenyl is optionally substituted with one or moregroups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen,C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ isC₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted witha group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is—C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen,C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy,C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyloptionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyloptionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy;or R₁₀ and R₁₁ together with the nitrogen to which they are attached isazetidine, aziridine, pyrrolidine, piperidine, homopiperidine,homopiperazine, morpholine or thiomorpholine, in which each isoptionally substituted with a group selected from halogen, C₁₋₆alkyl andC₁₋₄alkoxy; m is 0 or 1; or a nontoxic pharmaceutically acceptable saltthereof.
 4. A compound of Formula (IV);

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substitutedwith cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉;R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, benzyloxy, phenyl orCF₃ in which said phenyl is optionally substituted with one or moregroups selected from halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen,C₁₋₆alkyl, C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ isC₁₋₆alkyl or C₃₋₆cycloalkyl in which each is optionally substituted witha group selected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is—C₁₋₆alkylNR₁₀R₁₁; R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen,C₁₋₆alkyl optionally substituted with OH, halogen, C₁₋₄alkoxy,C₃₋₆cycloalkyl, thienyl or C₁₋₄alkylcarboxyl, —(CH₂)_(m)C₃₋₆cycloalkyloptionally substituted with phenyl or C₁₋₄alkyl; —(CH₂)_(m)phenyloptionally substituted with halogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy;or R₁₀ and R₁₁ together with the nitrogen to which they are attached isazetidine, aziridine, pyrrolidine, piperidine, homopiperidine,homopiperazine, morpholine or thiomorpholine, in which each isoptionally substituted with a group selected from halogen, C₁₋₆alkyl andC₁₋₄alkoxy; m is 0 or 1; or a nontoxic pharmaceutically acceptable saltthereof.
 5. A compound of Formula (IV);

wherein R₁ is phenyl optionally substituted with one or more groupsselected from halogen, C₃₋₆cycloalkyl, C₁₋₆alkyl optionally substitutedwith cyclopropyl, or —(CH₂)_(m)—, —(CH₂)_(m)—NHC(═O)R₈ and —NHC(═O)R₉;R₄ is hydrogen, C₁₋₆alkyl, C₁₋₄alkoxy, benzyloxy, phenyl or CF₃ in whichsaid phenyl is optionally substituted with one or more groups selectedfrom halogen, CN, CF₃ and OCH₃; R₇ is hydrogen, halogen, C₁₋₆alkyl,C₁₋₄alkoxy, CN, OH, NH₂, N(CH₃)₂, NO₂, or CF₃; R₈ is C₁₋₆alkyl orC₃₋₆cycloalkyl in which each is optionally substituted with a groupselected from halogen, CN, CF₃ and C₁₋₄alkoxy; R₉ is —C₁₋₆alkylNR₁₀R₁₁;R₁₀ is hydrogen or C₁₋₆alkyl; and R₁₁ is hydrogen, C₁₋₆alkyl optionallysubstituted with OH, halogen, C₁₋₄alkoxy, C₃₋₆cycloalkyl,—(CH₂)_(m)C₃₋₆cycloalkyl; —(CH₂)_(m)phenyl optionally substituted withhalogen, hydroxyl, C₁₋₄alkyl or C₁₋₄alkoxy; or R₁₀ and R₁₁ together withthe nitrogen to which they are attached is azetidine, aziridine,pyrrolidine, piperidine, homopiperidine, homopiperazine, morpholine orthiomorpholine; m is 0 or 1; or a nontoxic pharmaceutically acceptablesalt thereof.
 6. A pharmaceutical composition comprising atherapeutically effective amount of a compound of claim 1 in associationwith a pharmaceutically acceptable adjuvant, carrier or diluent.
 7. Amethod for the treatment of cerebral amyloid angiopathy and Down'sSyndrome in a mammal having cerebral amyloid angiopathy or Down'sSyndrome, which comprises administering to a mammal in need thereof atherapeutically effective amount of a compound of claim 1.